EXPRESSION AND CHARACTERIZATION OF A TRUNCATED, SOLUBLE, LOW-DENSITY-LIPOPROTEIN RECEPTOR

The low-density lipoprotein (LDL) receptor mediates the clearance of a polipoprotein B- and E-containing lipoproteins from the bloodstream. I n the current study, we characterized the binding properties of the am ino terminus of the LDL receptor. We produced a recombinant baculoviru s that encoded the first 354 amino acids, including the endogenous sig nal sequence, of the human LDL receptor. This truncated receptor prote in (LDL-R(354)) was secreted from recombinant baculovirus-infected Spo doptera frugiperda (Sf-21) cells. Upon electrophoresis, LDL-R(354) mig rated with a mobility of 55,000. Treatment of cells with tunicamycin d ecreased the size of the truncated receptor, suggesting the presence o f asparagine-linked carbohydrates. Nonreducing SDS-PAGE resulted in at least three discernible bands with M(r)s consistent with the truncate d receptor existing as monomers and multimers, suggesting the possibil ity of intermolecular disulfide cross-linking. All forms of the trunca ted receptor bound LDL on a ligand blot in a calcium-dependent manner. The purified truncated receptor bound I-125-LDL with high affinity in a competitive binding assay (IC50 = 0.8 mu g/ml). LDL-R(354) also bou nd calcium. This interaction was sensitive to the conformation of the ligand binding domain, as reduction of the disulfide bonds greatly dec reased the affinity of the receptor for calcium. The ligand and calciu m binding activities of this truncated receptor protein demonstrate th at the ligand binding domain of the LDL receptor can fold into a funct ionally active protein. (C) 1996 Academic Press, Inc.